The long-term objective of this project has been to investigate the roles of viral DNA replication, replication proteins, and gene expression in the interaction of herpes simplex virus (HSV) with the mammalian nervous system, especially virus latency. Latency is the most fascinating biological property of the virus and its most important clinical feature. Understanding HSV latency is sure to reveal novel features of HSV and the nervous system. Such information is highly relevant to antiviral drugs that target DNA replication, vaccines, and potential agents to cure HSV infections. The proposed research will investigate regulation of viral gene expression during latency (aim 1). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays will be used to measure expression of different classes of transcripts and potential regulatory molecules in ganglia and single cells. Cloning of cDNAs will complement the RT-PCR studies. These assays, mutant viruses from the Schaffer and Knipe laboratories, and transgenic mice overexpressing viral sequences will be used to study mechanisms of repression mediated by the HSV latency-associated transcript locus. Anti-HSV drugs will be used to test a model for regulation of viral gene expression by DNA replication. The mechanisms by which drug resistant mutants retain pathogenicity will be explored (aim 2). Frameshift mutations in the thymidine kinase gene that arise in clinical isolates will be tested for effects on latency. Cosmid-based methods will be used to map alleles of clinical isolates that compensate for loss of TK in latency. Finally, changes in host gene expression, which may affect latency, will be studied in collaboration with the Knipe laboratory and Millennium Pharmaceuticals (aim 3). Array technologies will be used to identify changes in host gene expression and RT-PCR assays and viral mutants will be used to examine the time-course, localization, and requirements for these changes. The proposed experiments should shed light on viral and cellular factors involved in the establishment, maintenance, and reactivation of HSV latency.